TR&D3:BioprintingforComplexScaffoldFabrication ProjectSummary Most biological tissues are composed of a complex microarchitecture with well-defined extracellular matrix (ECM)composition,mechanicalproperties,andcellpopulations.Duetothishighlevelofcomplexity,wehave employedamulti-material3Dprintingsystemwiththepotentialtorecapitulateheterogeneoustissuebyprinting material composites and depositing growth factors in a gradient organization to address three Specific Aims: (1) printing polymer/ceramic composites (of poly(?-caprolactone) (PCL), poly(propylene fumarate) (PPF), hydroxyapatite (HA), and ?-tricalcium phosphate (?-TCP)), (2) controlling the deposition and release of bone morphogeneticprotein-2(BMP-2)andvascularendothelialgrowthfactor(VEGF)fromPPF-basedscaffoldsfor vascularized bone repair in a femoral defect, and (3) depositing insulin-like growth factor-1 (IGF-1) and transforming growth factor ?-1 (TGF-?1) in a gradient organization within the 3D printed (3DP) PCL-based scaffoldsforosteochondraltissuerepair.Therationalefortheuseofamulti-material3Dprintingsystemisthe abilitytoprintawidevarietyofmaterials(polymers,ceramics,andbioactivemolecules),theaccessibilityand flexibility of deposition systems that can be incorporated with the printer, and the ability to establish the parametric space for printing various polymers within the realm of open-source technologies. Due to the flexibility of this system to accommodate a variety of tissue types and constructions, a wide variety of collaborative and service projects are proposed to be beneficial for TR&D3. The versatility of the proposed multi-material printing system provides a platform to modify scaffold design criteria for different materials, bioactive molecules, or cell types. We offer expertise in the processing of polymers (PCL, PPF) and ceramic pastes (HA, ?-TCP) for extrusion-based printing and will work with the other TR&Ds to optimize accuracy of printingtheseandotherbiomaterialsforuseindynamicculturesystems(TR&D1)andcontrolledcellprinting (TR&D2).Toextendtherelevanceofourmulti-materialprintingtechnology,wewillutilizealternativematerials developed by the Collaborative Projects, such as injectable, guest-host hydrogels and native tissue-derived ECM components for the 3D printing of osteochondral scaffolds, as well as injectable, thermoresponsive polymers and alginates for the 3D printing of bone scaffolds. Through these interactions, we will offer our expertiseinfabricatinggradientheterogeneitieswithintheseconstructstodirectcellularandtissueresponses. We will also provide technologies for the creation of scaffolds with growth factor gradients for applications in therepairoftissueswithzonalorganizationaswellaslargeskeletaldefects.